CN108925549B

CN108925549B - Cell freezing and storing carrying rod and preparation method and application thereof

Info

Publication number: CN108925549B
Application number: CN201810907123.1A
Authority: CN
Inventors: 黄斌; 陈艳
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2018-08-10
Filing date: 2018-08-10
Publication date: 2021-03-26
Anticipated expiration: 2038-08-10
Also published as: CN108925549A

Abstract

The embodiment of the invention provides a cell freezing and loading rod, which comprises a rod handle part and a cell attachment part connected with the rod handle part, wherein the cell attachment part comprises a first surface, at least one trapping groove is arranged on the first surface, and a hydrophobic region is arranged on the first surface and surrounds the trapping groove. The cell freezing and storing rod can effectively avoid the loss of the biological sample, effectively protect the biological sample from being damaged by the ice crystals of residual liquid, and improve the survival rate and activity of the biological sample. The invention also provides a preparation method and application of the cell cryopreservation carrying rod.

Description

Cell freezing and storing carrying rod and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological material cryopreservation, in particular to a cell cryopreservation carrying rod and a preparation method and application thereof.

Background

In recent years, the incidence of infertility is on the rise, and the in vitro assisted reproduction technology is very important. The most important link of the in vitro reproductive technology is embryo or egg cell cryopreservation. At present, the vitrification freezing technology is widely adopted, has the advantages of high freezing speed, ice crystal formation avoidance, cold melting damage reduction, simple operation and the like, and has higher survival rate after recovery and pregnancy success rate. At present, vitrification refrigerating fluid used in vitrification freezing technology mainly comprises base fluid, equilibrium fluid and vitrification fluid, and an embryo biological sample can enter liquid nitrogen after being subjected to vitrification freezing protection sequentially through the above fluids, and is frozen rapidly and kept for a long time. Carriers that have been commonly used in the art of vitrification freezing so far include cryotubes, open elongated tubules, copper mesh for electron microscopy, cryorings (Cryoloop), aluminum foils, and carrier rods, etc. The carrying rod is commonly used for open cryopreservation, namely cryopreservation of embryos and other samples by directly contacting liquid nitrogen, and has the advantages of fast cooling, obvious freezing protection effect and the like; simultaneously, when thawing recovery, only need to carry the pole to take out from the liquid nitrogen, alright carry out the operation of thawing immediately, it is more convenient to compare in other carriers.

However, the surface of the existing carrying rod is smooth, when a small drop of biological samples such as embryos or ova is dropped on the carrying rod during freezing, the biological samples are not stably adsorbed, and the biological samples are easily lost during operation. Meanwhile, excessive liquid (such as vitrification liquid) is easily remained on the existing carrying rod, so that in the subsequent freezing and storing process, the liquid easily influences the cooling rate of the biological sample and has great influence on the biological activity of thawing and reviving the biological sample; and the ice crystals formed by the liquid are prone to physical damage to the biological sample. Therefore, there is a need to develop a convenient and practical carrying rod capable of effectively preventing the loss of the biological sample and improving the survival rate of the biological sample.

Disclosure of Invention

In view of this, the embodiment of the present invention provides a cell freezing and loading rod, and a preparation method and an application thereof, wherein the cell freezing and loading rod can effectively avoid loss of a biological sample, effectively protect the biological sample from being damaged by ice crystals of residual liquid, and improve the recovery survival rate of the biological sample.

In a first aspect, the invention provides a cell freezing and loading rod, which comprises a rod handle part and a cell attachment part connected with the rod handle part, wherein the cell attachment part comprises a first surface, at least one trapping groove is arranged on the first surface, and a hydrophobic region is arranged on the first surface and surrounds the trapping groove.

Optionally, the hydrophobic region comprises a plurality of protrusions arranged in an array on the first surface.

Optionally, the center-to-center distance between two adjacent protrusions is 40-80 μm; the cross-sectional diameter of each of the protrusions is 10 to 40 μm.

Optionally, the cross-sectional diameter of the trapping groove is 100-230 μm; the depth of the trapping groove is 100-200 mu m. The trapping groove can be used for trapping biological samples such as embryonic cells, ova or other cells and the like, and can effectively prevent the biological samples from being lost; simultaneously, imprisoning is in the biological sample of imprisoning in the recess has avoided whole cryopreserving in-process to relapse through the process that shifts, and the time of cryopreserving operation significantly reduces improves the efficiency of cryopreserving operation.

Optionally, the protrusions comprise one or both of cylindrical and prismatic shapes.

Optionally, the hydrophobic region comprises a layer of hydrophobic material coated on the first surface.

Optionally, the biological sample comprises one or more of an embryo and an egg cell. The biological sample also includes tissue cells or tissues. Preferably, the biological sample comprises one or more of an embryo and an egg cell.

The cell freezing and loading rod provided by the first aspect of the invention is provided with the trapping groove, so that a target biological sample can be effectively trapped and locked; the cell freezing and storing device is provided with the hydrophobic area with good effect, the hydrophobic area can effectively reduce the redundant liquid of the biological sample on the cell attachment part of the cell freezing and storing carrying rod, so that the biological sample in the trapping groove can avoid the damage of the redundant liquid to the biological sample as far as possible, a faster cooling rate is maintained in the whole cell freezing process, and the recovery survival rate and the biological activity of the biological sample are greatly improved.

In a second aspect, the invention provides a method for preparing a cell freezing and storing rod, which comprises the step of preparing the cell freezing and storing rod by adopting a hot stamping method or a casting and forming method.

Optionally, the process of preparing the cell cryopreservation rod by using a hot stamping method includes:

providing a hard substrate, and forming at least one trapping groove and a plurality of protrusions arranged around the trapping groove on one side surface of the hard substrate to obtain a hard template with the trapping groove and the protrusions;

after the surface of the hard template is treated, pouring the hard template by using a mold adhesive, and after vacuum drying and curing treatment, uncovering a film to obtain an imprinting template, wherein the imprinting template comprises a pattern structure matched with the trapping groove and the bulge;

and providing a carrying rod, wherein the carrying rod comprises a rod handle part and an extension part connected with the rod handle part, and hot stamping is carried out by using the stamping template to obtain the cell freezing and storing rod.

Optionally, the drying time of the vacuum drying is 5-30 minutes; the curing temperature of the curing treatment is 75-90 ℃, and the curing time is 1-5 hours.

Optionally, etching the hard substrate by any one of an electron beam etching method, a photolithography method and a wet etching method to obtain the trapping groove and the hard template of the hydrophobic region.

Optionally, the mold paste includes one of Polydimethylsiloxane (PDMS), Polymethylmethacrylate (PMMA), Ethylene Vinyl Acetate (EVA), and Polyurethane (PUA). Preferably, the mold paste comprises polydimethylsiloxane. Alternatively, the preparation of the polydimethylsiloxane comprises: and mixing the silicon resin and the curing agent according to the mass ratio of (8-15) to 1, and uniformly stirring to obtain the polydimethylsiloxane.

The preparation method of the cell freezing carrying rod in the second aspect of the invention has simple process and low cost, and can be used for large-scale industrial production; the cell freezing and storing rod prepared by the preparation method can effectively avoid the loss of the biological sample, effectively protect the biological sample from being damaged by the ice crystals of residual liquid, and improve the survival rate and the biological activity of the recovered biological sample.

In a third aspect, the present invention also provides a cryopreservation apparatus comprising the cell cryopreservation rod according to the first aspect of the present invention or the cell cryopreservation rod obtained by the preparation method according to the second aspect of the present invention.

Optionally, the cryopreservation device further comprises a carrying rod bracket or an intelligent information processor. The carrying rod support is detachably connected with the rod handle part of the cell cryopreservation carrying rod. The carrying rod support can be used for fixing and neatly arranging a plurality of cell freezing carrying rods. Optionally, the intelligent information processor may be configured to record the biological sample information of each cell cryopreservation rod and the position information of each cell cryopreservation rod in the whole cryopreservation apparatus at any time. The intelligent information processor may be used to facilitate management of biological samples.

The cryopreservation device of the third aspect of the invention can be used for efficiently and safely performing cryopreservation and management on a biological sample. For example, the cryopreservation device can be an embryo vitrification cryopreservation device.

Advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of embodiments of the invention.

Drawings

In order to more clearly illustrate the contents of the present invention, a detailed description thereof will be given below with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a schematic structural diagram of a cell freezing bar 100 according to an embodiment of the present invention;

FIG. 2 is a schematic structural view of a cell freezing bar according to another embodiment of the present invention;

FIG. 3 is a schematic structural view of a cell attaching part of a cell freezing and loading rod according to an embodiment of the present invention;

FIG. 4 is a schematic side view of the cell attaching part of the cell freezing bar according to one embodiment of the present invention.

FIG. 5 is a diagram illustrating an actual effect test of the hydrophobic region according to an embodiment of the present invention;

fig. 6 is a process flow chart of a method for preparing a cell freezing bar according to an embodiment of the present invention.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it should be noted that those skilled in the art can make various modifications and improvements without departing from the principle of the embodiments of the present invention, and such modifications and improvements are considered to be within the scope of the embodiments of the present invention.

The terms "comprising" and "having," and any variations thereof, as appearing in the specification, claims and drawings of this application, are intended to cover non-exclusive inclusions. For example, a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to only those steps or elements listed, but may alternatively include other steps or elements not listed, or inherent to such process, method, article, or apparatus.

Unless otherwise specified, the raw materials and other chemicals used in the examples of the present invention are commercially available.

As shown in FIG. 1, one embodiment of the present invention provides a cell freezing and loading rod 100, which comprises a rod handle 10 and a cell attaching portion 20 connected to the rod handle 10, wherein the cell attaching portion 20 comprises a first surface 21, at least one trapping groove 201 is formed on the first surface 21, and a hydrophobic region 202 is formed on the first surface 21 and surrounds the trapping groove 201.

In this embodiment, referring to fig. 1, in the cell freezing and storing rod 100, the rod handle portion 10 and the cell attaching portion 20 are each in a quadrangular prism shape, and the structure inside the dotted line frame in the figure is the cell attaching portion 20 of the cell freezing and storing rod 100; the cell attachment portion 20 includes at least one surface, namely a first surface; wherein the first surface 21 is in the shape of a flat rectangle.

Alternatively, the stem part 10 and the cell attaching part 20 may also be different other shapes, respectively. For example, the stem handle 10 may include, but is not limited to, a round rod shape, or a semi-round rod shape, a polygonal prism shape, or other shapes. The interface shape of the cell attachment portion 20 may include, but is not limited to, a parallelogram, a trapezoid, or other shapes. For example, the interface shape of the cell attaching part 20 is a quadrangle in which two sides are parallel and the other two sides are curved. Alternatively, the shape of the first surface 21 may be, but is not limited to, a planar rectangular shape. The shape of the first surface 21 may also be a planar circle, a planar trapezoid or other shapes.

In this embodiment, the hydrophobic region 202 is a circular region centered on the trapping groove 201, as shown in FIG. 1. Alternatively, the hydrophobic area 202 may be other shaped areas, such as a rectangle, a triangle, a pentagon, a hexagon, etc., or may be irregular.

Alternatively, the outer lateral side edge of the hydrophobic region 202 can be, but is not limited to, a fixed value of the distance from the opening edge of the trapping groove 201; namely, the area of the hydrophobic region 202 arranged around each of the trapping grooves 201 is constant. For example, the distance between the peripheral edge of the hydrophobic region 202 and the opening edge of the trapping groove 201 is 500-. Still further, the hydrophobic region may also cover the entire first surface. For example, referring to FIG. 2, on the first surface 21, the hydrophobic region 202' covers the other areas except the trapping groove 201. In this embodiment, by adjusting the distance between the edge of the outer side surface of the hydrophobic region and the edge of the opening of the trapping groove, the area of the hydrophobic region can also be changed; the hydrophobic extent of the hydrophobic region varies for different areas.

In this embodiment, the hydrophobic region 202 may further include, but is not limited to, a plurality of protrusions arranged in an array on the first surface 21. Optionally, the hydrophobic region 202 may also include, but is not limited to, a plurality of protrusions 203 arranged in a vertical array on the first surface 21; the protrusions 203 are integrally formed with the cell attaching part 20, see fig. 3. Alternatively, the protrusions may include, but are not limited to, one or both of a cylinder and a prism. For example, the protrusions are in the shape of a cylinder, or the shape of a triangular prism, or the shape of a quadrangular prism, or the shape of a pentagonal prism, or the shape of another polygonal prism. Optionally, the included angle between each protrusion and the first surface can also be an acute angle or an obtuse angle; i.e. the protrusions are arranged non-perpendicularly on the first surface. Optionally, the protrusion is integrally formed with the cell attachment portion.

Further, as shown in fig. 4, the center-to-center distance between two adjacent protrusions 203 is L; each of the protrusions 203 has a cross-sectional diameter D. The center-to-center distance refers to the distance between the centers of two adjacent projections. Optionally, the center-to-center distance L between two adjacent protrusions is 40-80 μm. Further, optionally, the center-to-center distance L between two adjacent protrusions is 45-70 μm. For example, the center-to-center distance L between two adjacent protrusions is 40 μm, or 50 μm, 60 μm, 70 μm, and 80 μm. Optionally, the cross-sectional diameter D of each of the protrusions is 10-40 μm. Further, optionally, the cross-sectional diameter D of each of the protrusions is 15 to 40 μm. For example, the cross-sectional diameter D of each of the projections is 10 μm, or 20 μm, or 25 μm, or 30 μm, or 40 μm. The hydrophobic effect of the hydrophobic region can be adjusted by adjusting the center-to-center distance or the cross-sectional diameter of the projections.

Optionally, the height h of each of said protrusions is 20-60 μm, see fig. 4. Further, optionally, the height h of each protrusion is 25-50 μm. For example, the height h of each of the protrusions is 20 μm, or 25 μm, 30 μm, 40 μm, 50 μm, 60 μm.

Optionally, each of the protrusions is further provided with a plurality of micro-convex structures; the micro-convex structures include one or more of cylindrical, conical, and prismatic. Optionally, the distance between centers of two adjacent micro-convex structures is 2-5 μm. The diameter of the micro-convex structure attached to the convex surface is 0.1-1 μm. Further, optionally, when the cross-sectional height of the micro-convex structure is 0.5-2 μm. The micro-convex structure can further improve the hydrophobic effect of the hydrophobic area. When the trapping groove of the cell freezing and carrying rod is surrounded by the hydrophobic region with the microprotrusion structure, the cell freezing and carrying rod has a better freezing and carrying effect in the process of freezing and carrying out biological samples.

Optionally, the cross-sectional shape of said trapping groove 201 is circular, see fig. 4; the cross-sectional diameter of the trapping groove 201 is R; the depth of the trapping groove 201 is H. Alternatively, the cross-sectional shape of the trapping groove can be, but is not limited to, one or more of circular, rectangular, or triangular. Further, optionally, the cross-sectional shape of the trapping groove also comprises other polygons. For example, the cross-sectional shape of the trapping groove is triangular, pentagonal or hexagonal. When the cell freezing bar according to this embodiment includes a plurality of the trapping grooves, the cross-sectional shapes of the plurality of trapping grooves may be the same or different. For example, the cross-sectional shapes of the plurality of trapping grooves can include two or more of circular, rectangular, and triangular shapes at the same time.

Optionally, the radius of curvature of the inner wall of the trapping groove gradually decreases from the opening of the trapping groove to the bottom of the trapping groove. In this embodiment, inner wall radius of curvature follows imprisoning groove opening extremely imprisoning groove bottom reduces gradually imprisoning groove laminates biological sample to a certain extent more, both can effectively imprison biological sample at the cryopreserving in-process, can further be less again between biological sample the gathering of unnecessary liquid on every side, and whole cell is kept a faster cooling rate in the freezing process, improves biological sample's recovery survival rate and biological activity greatly.

In this embodiment, the inner wall surface of the trapping groove may further include, but is not limited to, a layer of second hydrophobic region, where the second hydrophobic region includes a second hydrophobic material layer coated on the inner wall surface or a plurality of second protrusion structures arranged in a vertical array on the inner wall surface. Optionally, the cross-sectional diameter of the second projection structure of the inner wall surface of the trapping groove is 10 to 40 μm. Optionally, the center-to-center distance between adjacent second bump structures is 30-50 μm. The height of the second bump structure is 30-50 μm. Optionally, the second hydrophobic region corresponding to the inner wall surface of the trapping groove and the hydrophobic region of the trapping groove around the first surface may be the same or different. Alternatively, the plurality of second projection structures can be, but are not limited to being, integrally formed with the trapping groove.

The second hydrophobic material layer and the second protruding structures can enable the inner walls of the trapping grooves to have good hydrophobic performance. When the inner wall surface of the trapping groove has hydrophobicity, the liquid wrapping the cells can be greatly reduced, the total volume of the liquid drops is smaller, and the cooling is faster. Further, the biological sample maintains a more balanced and rapid cooling state in the whole cell freezing process, so that the resuscitation survival rate and the biological activity of the biological sample are greatly improved, and the freezing effect of the cell freezing carrying rod is further improved.

Further, optionally, when the cross-sectional shape of the trapping groove 201 is circular, the cross-sectional diameter R of the trapping groove 201 is the diameter of the circular cross-sectional shape; when the cross-sectional shape of the trapping groove 201 is non-circular, the cross-sectional diameter R of the trapping groove 201 is the diameter of an inscribed circle of the non-circular cross-sectional shape. Optionally, the cross-sectional diameter of the trapping groove is 100-230 μm. Further, optionally, the cross-sectional diameter of the trapping groove is 120-. For example, the cross-sectional diameter of the trapping groove is 100 μm, or 120 μm, or 150 μm, or 180 μm, or 200 μm, or 230 μm. Optionally, the depth H of the trapping groove is 100-. Further, optionally, the depth H of the trapping groove is 120-. For example, the depth H of the trapping groove is 100 μm, or 120 μm, or 150 μm, or 170 μm, or 180 μm, or 200 μm. In this embodiment, the trapping groove with the depth and/or the size of the cross-sectional diameter is matched with the size of a biological sample including an embryo or an egg cell, so that the biological sample such as an embryo or an egg cell can be better trapped, the liquid content of the biological sample can be scientifically maintained while the biological sample is prevented from being lost, the adhesion of redundant liquid to the biological sample is reduced, the damage of the redundant liquid to the biological sample in the trapping groove is avoided as much as possible, a faster cooling rate is maintained in the whole cell freezing process, the recovery survival rate and the biological activity of the biological sample are greatly improved, and the cost is saved. By adjusting the depth of the trapping groove and/or the size of the diameter of the cross section, the cell freezing and loading rod can also be used for freezing and storing biological samples including stem cells and the like.

The hydrophobic region structure comprising a plurality of protrusions according to the embodiment of the present invention has a good hydrophobic effect, as shown in fig. 5, the hydrophobic region can effectively make a contact angle between a liquid drop loaded on the surface of the hydrophobic region and the surface of the hydrophobic region become extremely large, so that a rolling angle between the liquid drop and the surface of the hydrophobic region is small; the cell freezing carrying rod is effectively prevented from carrying redundant liquid in the process of freezing the biological sample, the freezing effect of the biological sample is effectively ensured, and the follow-up bioactivity of the biological sample after recovery is improved. The volume of the excess liquid can also be adjusted to a certain extent by providing a hydrophobic region surrounding the trapping groove.

In this embodiment, the trapping groove can be provided at any position on the first surface. Alternatively, the trapping groove can be, but is not limited to, an intermediate site provided on the cell attaching portion. Alternatively, the trapping groove may be provided on the cell attaching portion at a position near the peripheral edge of the cell attaching portion. Optionally, the cell attachment portion can be provided with, but not limited to, two or more trapping grooves; the two or more trapping grooves are at any position on the same side surface on the cell attaching portion. For example, when a plurality of the trapping grooves are provided on the cell attaching portion, the plurality of trapping grooves can be uniformly distributed on the same side surface of the cell attaching portion.

In this embodiment, when the hydrophobic region is a structure including a plurality of protrusions, the trapping groove and the hydrophobic region are both disposed on the first plane; therefore, the plane of the opening of the trapping groove is not located on the same interface with the surface of the side of the hydrophobic region away from the first surface.

In another embodiment of the present invention, the hydrophobic region on the first surface surrounding the trapping groove can include, but is not limited to, a layer of hydrophobic material applied to the first surface. The hydrophobic material layer has good hydrophobic ability. Optionally, the material of the hydrophobic material layer may include, but is not limited to, a hydrophobic coating with low temperature resistance and stable structure.

In this embodiment, the cross-sectional thicknesses of the cell attaching part and the stem part may be the same or different; the cell attaching part and the stem part are of an integral structure.

In this embodiment, a marking region may be further provided on a top surface or a side surface of the port of the stem portion of the cell freezing and loading rod, the port being located at a side away from the cell attaching portion, and the marking region may be used to fill in the freezing and storing information. For example, the label area may be filled with freezing time, biological sample information, and the like. The marker region may also be used to distinguish each of the cell cryopreserved rods to some extent.

In the prior art, embryos or other biological samples are usually directly placed on the freezing and storing carrying rod, and the embryos are small, the freezing and storing process is rapid, so that the biological samples can be rapidly cooled and frozen on the freezing and storing carrying rod after entering liquid nitrogen. However, this method is often accompanied by a large amount of liquid and is frozen on the freezing and freezing rod, and the freezing and freezing method is not firm, and when the size of the biological sample is large, the biological sample is more likely to fall off and lose, and the waste of liquid is also caused. The cell freezing and loading rod provided by the embodiment of the invention is provided with the trapping groove, so that a target biological sample can be effectively trapped and locked; the cell freezing and storing device is provided with a hydrophobic area with a good effect, the hydrophobic area can effectively reduce redundant liquid on a cell attachment part of the cell freezing and storing rod, so that the biological sample in the trapping groove is prevented from being damaged by the redundant liquid as much as possible, a faster cooling rate is maintained in the whole cell freezing process, and the recovery survival rate and the biological activity of the biological sample are greatly improved.

The embodiment of the invention provides a preparation method of a cell freezing and carrying rod, which comprises the step of preparing the cell freezing and carrying rod by adopting a hot stamping method or a casting and forming method.

Alternatively, as shown in FIG. 6, an embodiment of the present invention provides a method for preparing the cell cryopreservation rod by using a hot stamping method, comprising

S10, providing a hard substrate, and forming at least one trapping groove and a plurality of protrusions arranged around the trapping groove on one side surface of the hard substrate to obtain a hard template with the trapping groove and the protrusions;

s20, after surface treatment is carried out on the hard template, the hard template is poured by using model glue, after vacuum drying and curing treatment, a film is uncovered, and an imprinting template is obtained, wherein the imprinting template comprises a pattern structure matched with the trapping groove and the bulge;

and S30, providing a carrying rod, wherein the carrying rod comprises a rod handle part and an extension part connected with the rod handle part, and hot stamping is carried out by using the stamping template to obtain the cell freezing carrying rod.

Specifically, in step S10, the material of the hard substrate includes hard metal or hard high molecular polymer. Optionally, the hard metal may include, but is not limited to, one or more of tungsten, molybdenum, iron, nickel, vanadium, titanium, and copper. Further, optionally, the hard metal further comprises a hard alloy comprising one or more of tungsten, molybdenum, iron, nickel, vanadium, titanium, and copper. For example, the hard metal is nickel-iron alloy, or ferromolybdenum alloy, or ferrotungsten alloy, or titanium-copper alloy, etc. The hard high molecular polymer comprises one or more of polymethyl methacrylate (PMMA), Polycarbonate (PC), Cyclic Olefin Copolymer (COC), Polystyrene (PS), Polyoxymethylene Oxide (POM) and acrylonitrile-butadiene-styrene copolymer (ABS).

Optionally, the hard substrate is etched by any one of an electron beam etching method, a photolithography method and a wet etching method, so that the trapping groove and the plurality of protrusions arranged around the trapping groove are formed on one side surface of the hard substrate.

Specifically, in step S20, the surface treatment includes a silylation treatment. Optionally, the silanization treatment process comprises surface treatment of the hard template with a silanization reagent. Optionally, the silylating agent comprises a fluoroalkylsilane agent, or the like. Further, optionally, after obtaining the hard template with the trapping grooves and the protrusions, before the surface treatment, the hard template may be subjected to an activation treatment; the activation treatment process comprises the step of activating the hard template by adopting plasma or corona technology.

Optionally, the mold paste includes one of Polydimethylsiloxane (PDMS), Polymethylmethacrylate (PMMA), Ethylene Vinyl Acetate (EVA), and Polyurethane (PUA). Preferably, the mold paste comprises polydimethylsiloxane. Alternatively, the preparation of the polydimethylsiloxane comprises: and mixing the silicon resin and the curing agent according to the mass ratio of (8-15) to 1, and uniformly stirring to obtain the polydimethylsiloxane. Further optionally, the mass ratio of the silicone resin to the curing agent is (8-12): 1.

Optionally, the drying time of the vacuum drying is 5-30 minutes. Further, optionally, the drying time of the vacuum drying is 10-20 minutes. For example, the drying time of the vacuum drying is 5 minutes, or 10 minutes, or 15 minutes, or 20 minutes. The vacuum state is in a range of pressure lower than 110 Pa. Optionally, the curing temperature of the curing treatment is 75-90 ℃, and the curing time is 1-5 hours. Further, optionally, the curing temperature of the curing treatment is 75-85 ℃, and the curing time is 1-3 hours.

In this embodiment, the vacuum drying and curing process may remove gas in the mold compound, thereby effectively preventing the mold compound from generating bubbles in the cured imprint template; meanwhile, the stamping template is promoted to form a more stable structure, so that the stamping template has longer service life and durable characteristic. The activation treatment can improve the treatment effect of surface treatment such as silanization treatment of the hard template, improve the bonding force between the hard template and the hydrophobic silanization reagent molecules, and promote the hard template to better and more comprehensively complete the surface treatment process. The trapping grooves and the protrusions are of fine microstructures; therefore, the surface treatment process and the subsequent preparation of the imprinting template have an important influence on the quality of the cell cryopreservation rod; the surface treatment process and the imprinting template preparation process can greatly improve the yield of the cell cryopreservation carrying rod, so that the cell cryopreservation carrying rod has better capability of storing biological samples.

Specifically, in step S30, the imprint template is used to perform hot stamping on the extension portion, so that the trapping grooves and the protrusions are formed on the carrier bar. The carrier rod can be directly prepared by a conventional die forming method. In this embodiment, the imprint template can be used repeatedly.

The preparation method of the cell freezing and storing carrier rod provided by the embodiment of the invention has the advantages of simple process and low cost, and can be used for large-scale industrial production; the cell freezing carrying rod prepared by the preparation method can effectively avoid the loss of the biological sample, effectively protect the biological sample from being damaged by the ice crystals of residual liquid, improve the freezing effect of the biological sample and the bioactivity of the biological sample after recovery.

Optionally, an embodiment of the present invention further provides a method for preparing the cell cryopreservation rod by using a hot stamping method, including

S210, providing a hard substrate, and forming at least one trapping groove structure on one side surface of the hard substrate to obtain a hard template with the trapping groove structure;

s220, after surface treatment is carried out on the hard template, the hard template is poured by using model glue, after vacuum drying and curing treatment, a film is uncovered, and an imprinting template is obtained and comprises a pattern structure matched with the trapping groove structure;

and S230, providing a carrying rod, wherein the carrying rod comprises a rod handle part and an extension part connected with the rod handle part, and coating a hydrophobic material to form a hydrophobic area after hot stamping is carried out by using the stamping template, so that the cell freezing and storing carrying rod is obtained.

In the preparation method according to the embodiment of the invention, the hydrophobic region is a hydrophobic material layer, and the cell cryopreservation carrier rod obtained by the preparation method has a good biological sample cryopreservation effect and can be efficiently used for cryopreservation of a biological sample.

The invention also provides a method for preparing the cell freezing and storing rod by adopting a casting molding method, which comprises the following steps:

s310, providing a substrate;

s320, etching the substrate to form a reverse mold, wherein the reverse mold comprises a reverse structure of the stem part and the cell attachment part;

s330, pouring a cold-resistant material into the reverse mold, and separating the cold-resistant material from the reverse mold after molding treatment to obtain the cell freezing and carrying rod.

Specifically, in step S310, the material of the substrate may include, but is not limited to, one or more of tungsten, molybdenum, iron, nickel, vanadium, titanium, and copper. Further, optionally, the material of the substrate further includes a hard alloy containing one or more of tungsten, molybdenum, iron, nickel, vanadium, titanium, and copper. For example, the substrate is made of a nickel-iron alloy, a molybdenum-nickel-iron alloy, a tungsten-iron alloy, a titanium-copper alloy, or the like.

Specifically, in step S320, the etching the substrate to form the reverse mold includes etching the substrate by one or more of photolithography, wet etching, or dry etching to form the reverse mold. For example, a photoresist material is coated on the substrate by a mask method, and a reverse mold of the cell freezing and loading rod having the trapping groove and the hydrophobic region structure is formed on the substrate through patterning operations such as exposure, development, etching (Etch), and photoresist stripping. Optionally, the photoresist material comprises a resin, a photosensitizer and a solvent. The photoresist material may also include other materials, which are not limited in this embodiment.

Specifically, in step S330, since the cell cryopreservation bar is used for cryopreservation of a biological sample, the cell cryopreservation bar can withstand the temperature of liquid nitrogen. Alternatively, the cold-resistant material may be a metal, glass, or organic polymeric material. For example, the cold-resistant material is glass, steel, tetrafluoroethylene, polychlorotrifluoroethylene, or ultra-high molecular weight polyethylene.

The preparation method of the cell cryopreservation carrying rod provided by the embodiment of the invention adopts the mould to perform casting molding, can prepare the cell cryopreservation carrying rod in an integrated molding manner, has simple process and low cost, and can be used for large-scale industrial production.

In this embodiment, the size, shape and structure of the cell freezing and storing rod can also be adjusted by adjusting the process parameters of the preparation method provided by the embodiment of the present invention.

It should be noted that, according to the disclosure and the explanation of the above description, the person skilled in the art to which the present invention pertains may make variations and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some equivalent modifications and variations of the present invention should be covered by the protection scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. The utility model provides a cell cryopreserving carries pole, its characterized in that, including the pole stalk portion and with the cell adhesion portion that the pole stalk portion links to each other, cell adhesion portion includes the first surface, be equipped with at least one imprisoning groove on the first surface, just center on the first surface imprisoning groove is equipped with hydrophobic region, hydrophobic region includes that the array is arranged a plurality of archs on the first surface.

2. The cell freezing bar of claim 1, wherein the distance between the centers of two adjacent projections is 40-80 μm; the cross-sectional diameter of each of the protrusions is 10 to 40 μm.

3. The cell cryopreservation slide bar of claim 1, wherein the cross-sectional diameter of the trapping groove is 100-230 μm; the depth of the trapping groove is 100-200 mu m.

4. The cell freezing bar of claim 1, wherein said projections comprise one or both of cylindrical and prismatic shapes.

5. The cell cryopreservation bar of claim 1 wherein the hydrophobic region comprises a layer of hydrophobic material coated on the first surface.

6. The method according to claim 1, comprising preparing the cell freezing bar by hot stamping or cast molding.

7. The method according to claim 6, wherein the step of preparing the cell cryopreservation rods by hot stamping comprises:

8. The method according to claim 7, wherein the drying time of the vacuum drying is 5 to 30 minutes; the curing temperature of the curing treatment is 75-90 ℃, and the curing time is 1-5 hours.

9. A cryopreservation apparatus comprising the cell cryopreservation carrying rod of any one of claims 1 to 5.